The essential elements for substrate specificity for myosin I heavy chain kinase has been studied with the use of synthetic peptides as substrates. One basic amino acid has been found essential, and two preferable, on the N-terminal side of the phosphorylation site and a tyrosine reside is required 2 positions away on the C-terminal side. The activity of the kinase is about 50-fold enhanced by autophosphorylation of up to 8 sites and this autophosphorylation is about 20-fold stimulated by the presence of acidic phospholipids. Previous fluorescence immunolocalization studies of myosin I have been extended by immunoelectron microscopy. Myosin I has been found to be located at the plasma membrane, as previously inferred, and also at the membrane of the contractile vacuole, a new observation.